Acid and Alkaline Phosphatase Levels in GCF during Orthodontic Tooth Movement

Statement of the Problem The present constituents of gingival crevicular fluid (GCF) can reflect the changes occurring in underlying tissues. Considering variety of biologic bone markers, alkaline phosphatase and acid phosphatase have been examined as bone turn over markers in orthodontic tooth movement. Purpose The current study designed in a longitudinal pattern to determine the changes of acid and alkaline phosphatase (ACP & ALP) in GCF during orthodontic tooth movement. Materials and Method An upper canines from twelve patients (mean age: 14±2 years) undergoing extraction orthodontic treatment for distal movement served as the test tooth (DC), and its contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in orthodontic appliance without orthodontic force; the AC was free from any orthodontic appliance. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance placement (T0), and 14 (T2) and 28 days (T3) after it and ALP and ACP concentration were determined spectrophotometrically. Results ALP concentration was elevated significantly in DC and CC groups at days 14 and 28 compared with the AC. In DC group, the ALP was significantly greater in mesial sites than distal site, while no significant changes were found between both sites of CC. The peak level of ALP was observed in mesial sites of DC at T2. Regarding ACP, significant elevation of this enzyme was seen in DC group both in mesial and distal sites at T2 and T3. The peak level of this enzyme was seen at T2. Conclusion Monitoring simultaneous changes of ALP and ACP levels in GCF can reflect the tissue responses occur in periodontium during bone formation and bone resorption during orthodontic tooth movement, respectively.


Introduction
By increasing the number of patients seeking orthodontic treatment and correction of dentofacial deformities, investigations on biologic basis of orthodontic movement seem absolutely necessary. [1] Several investigations have evaluated the molecular basis of soft and hard tissue responses following orthodontic tooth movement. [2] By precise evaluation of the biologic response of orthodontic forces, the orthodontic force application could be based on each individual tissue responses and therefore the final effect of orthodontic treatment might be improved. [3] In addition, the main problem of retention can be solved to some extent by considering bone turnover rates around each experimental tooth. [3] However, providing a simple noninvasive method is required for achieving these possibilities. In recent years, a few constitutes of gingival crevicular fluid (GCF) have been shown to be diagnostic biomarkers of active tissue destruction in periodontal disease which might also serve as diagnostic markers of biologic responses in orthodontic tooth movement. [4][5] GCF is an osmotically mediated inflammatory exudate and its constituents are extracted from different sources including microbial dental plaque, inflammatory cells, host tissue, and particularly serum. [5] Many investigations have demonstrated that GCF constituents can be useful for determination of local biological and biomechanical processes associated with metabolic bone turnover during orthodontic tooth movement. [2,[6][7][8][9][10] Different assumptions have been suggested to explain the biological basis of orthodontic tooth movement. Recently, the studies suggest that the mechanical stimulants like orthodontic tooth movement may induce some subsequent inflammatory responses in periodontal tissue. [5,[11][12] The cells can release enough amounts of chemical mediators into GCF; therefore, the amount of these substances may increase during orthodontic tooth movement in GCF. [13] Considering variety of biologic bone markers, alkaline phosphatase (ALP) and acid phosphatase (ACP) have been examined as bone turn over markers in orthodontic tooth movement. [4-5, 12, 14-15] ALP is a glycoprotein thought to be involved in processes leading to mineral formation in tissue like bone and cementum. [15] Robinson (1923) was first in suggesting that the ALP is important in the mineralization of bone and calcifying cartilages. [16] The enzyme is thought to release phosphate ions from organic phosphate esters as a result of which supersaturation would occur, leading to the precipitation of calcium phosphate salts. [15] This enzyme is produced mainly by neutrophils at GCF, although it is also produced by variety of cells including fibroblasts, osteoblasts, and osteoclasts. [6] ACP is also an enzyme thought to be involved in processes of mineral deformation in tissues like bone.
Resorbing cells, such as osteoclasts and macrophages, have been shown to have high ACP activities. [14,17] Little information is available in the field of production of these chemical mediators during orthodontic tooth movement. For the first time, Keeling et al.
(1993) examined tartrate-resistance acid phosphatase (TRAP) changes in serum and alveolar bone during orthodontic tooth movement in 288 adult male rats. [14] The authors reported that enzyme activities increased during orthodontic tooth movement. In addition, high levels of ACP were detected on first day in serum and on third day in bone, whereas the peak of ALP activity in bone has occurred on seventh day. Insoft et al. (1996) also described the activity of both phosphatase on longitudinal study with three cases (as split mouth design) and on a cross-sectional design on 30 patients undergoing orthodontic treatment. [4] An increase in GCF ALP activity during orthodontic tooth movement occurred from 1 to 3 weeks of treatment and the peak of ACP activity occurred from 3 to 6 weeks. It is worth mentioning that this studying was the only investigations that have evaluated the ACP activity changes during orthodontic tooth movement in human subjects. [4] However, the design of the study was based on cross-sectional study without controls which is considered as the distinct methodological shortcoming; therefore, the achieved data could not be interpreted without cautions. [5] Parinetti et al. (2002) have examined ALP activity in gingival crevicular fluid during orthodontic tooth movement and demonstrated that the ALP activity was significantly higher in tension sites rather than compression sites of teeth undergoing orthodontic forces.
[5] Moreover, a positive correlation between GCF ALP activity and subgingival colonization of action bacillus actinomycetemcomitans (Aa) has been reported. [18] According to the restricted number of studies that have investigated the changes in levels of ALP and ACP activities during orthodontic tooth movement, the current longitudinal study was aimed to evaluate the both ALP and ACP activities changes in gingival crevicular fluid during orthodontic tooth movement. for using twice per day to minimize the possible role of gingival inflammation on changing enzymatic activ-ities. Before the beginning of the study, informed consents were obtained from all participants or guardians (for patients under 16 years of age).

Experimental and Control groups
Twelve patients (mean age: 14±2 years) after passing at least 7-10 days from the time of the extraction of the maxillary first premolar were participated in this study.
In each patient, maxillary canines underwent orthodontic treatment. One of the upper canines, the distalized canine (DC) was to be treated for distal movement and was used as the experimental tooth. The contralateral canine (CC) was received the orthodontic appliances but was not moved distally and thus used as  The data were statistically analyzed by means of repeated measures (ANOVA) and paired t-test using SPSS (Version 11). P value of <0.05 was considered as statistically significant.

Results
The clinical parameters of periodontal health of select-

Among time points
The GCF ALP activity was increased significantly at mesial sites of DC groups between the time points (T1-T2) and (T2-T3) (p< 0.001 for both time intervals). However, at distal sites of DC groups, this increasing trend was only seen from T1-T2 (p< 0.001).
The ALP activity decreased from T2-T3 which was not statistically significant (p= 0.163).
In contrast to T2-T3 time interval, the ALP activity changes at both mesial and distal sides of the CC groups were statistically significant between T1-T2 (p< 0.05). This enzyme activity at mesial and distal sites of the AC groups did not demonstrate a statistically significant change at any time interval (p= 0.145)

Different treatments
Similar to the data for GCF ALP, the ACP activity at baseline was the same among experimental groups at both mesial and distal sites (p= 0.83 and 0.915). As it is shown at Figure 4, a statistically significant change in ACP activity was seen at T2 in both mesial and distal sites of DC group compared with the mesial and distal site of CCs and AC groups, respectively (p< 0.05). However, this difference was not observed between ACP activity levels of both mesial and distal sites of three groups at T3 (p= 0.596 and 0.257).

Different sites
Result of the other statistical tests also did not show a significantly greater enzymatic activity in distal than mesial sites of the DC and AC groups at both the 14 th day (T2), and the 28 th day (p= 0.072 and 0.137, respectively) (Figure 4). Conversely, in the CC groups, a significant statistical difference in ACP activities was seen between the mesial and distal sites at T2 (p=0.05). However, this significant difference was not observed at two other time points (T1, and T3) (p= 0.298 and 0.068, respectively) ( Figure 4). The peak level of this enzyme was seen at the day 14. Moreover, in the AC group also, no significant statistical difference in ACP activities was seen between the mesial and distal sites at different time points (T1, T2 and T3) (p=0.157, 0.314 and 0.182, respectively) ( Figure 4).

Among time points
The GCF ACP activity was increased significantly at both mesial and distal sites of DC groups between the time points (T1-T2) (p< 0.05) and decreased thereafter on both sites (T2-T3). However, the reported changes within two time intervals were not statistically signifi-

Discussion
Our controlled longitudinal study has evaluated ALP and ACP GCF alterations at three different time points of orthodontic treatment. Although no differences were detected in the probe depth parameter between the groups, the plaque index and bleeding on probe index were greater in teeth receiving orthodontic appliance with or without force (CC and DC groups). Other studies have shown the similar result about gingival condition of teeth undergoing orthodontic treatment. [21][22] The underlying reason of observed differences could be the slight gingival enlargement following orthodontic treatment. Therefore, in studies evaluating the periodontal status of orthodontic patients, the site-specific characteristic of periodontal parameters and also the direct effect of reinforcement of oral hygiene instructions on periodontal condition of the teeth during orthodontic tre-atment should be considered. [23] ALP has been reported as biologic marker of osteoblastic activity during bone deposition. [24][25][26][27] It is well proven that after inserting an orthodontic force, an early phase (3-5 days) of non-specific bone resorption activity is followed by an early (5-7 days) and a late phase (7-14 days) of bone deposition. [5] In the current study, high level of ALP activity was also seen on the 14 and 28 days (T2 and T3) in both mesial and distal of DCs which is supported by the approved hypothesis that described increased osteoblastic activity could take place in both tension and compression sites in alveolar bone. [5,10,14,28] In contrast to controversial findings of animal studies evaluating ALP activity in periodontal ligament, the result of this study is in accordance with previous longitudinal studies on human subjects. [5,29] Some previous studies observed that the peak of ALP activity occurred at day 14 whereas in our study the peak was seen at the day 14 and 28 at mesial and distal sites of canine retraction groups, respectively. [5,9,26] Some other investigations also reported bone formation appeared to begin after osteoclastic resorption phase that could last from 10 days to 3 weeks. [28,30] These studies might therefore support the observed increase in observed difference can be attributed due to different force magnitude on teeth in different studies and also the limited duration of study. [10,14] Greater ALP activity, though not statistically significant, in the CC group than AC group can be attributed to several subclinical displacements and movements that exerted by the wire in first step of leveling and alignments [5,27] and also the dental plaque accumulation due to orthodontic appliance presence. [6,12,31] The greater ALP activity values reported for the mesial (tension) sites compared with distal sites (compression) at DC group in both T2 and T3 might be as a consequence of greater bone deposition process over the resorption process in tension site. [30] The results obtained in the present study, which are in agreement with other studies, suggest that the increase in ALP activity in GCF might be related to dental site bone remodeling following orthodontic forces. [4,14,[29][30] ACP also has been reported as a biologic marker of bone destruction. [4] Data from our study, as well as other researches, have shown that in the early phase of tooth movement, the ACP activity is greater than later phase that follows by increasing in the ALP activity and decreasing in the ACP activity. [4][5] In this study, in contrast to Insoft et al. investigation, [4] the peak of ACP concentration was observed at day 14 (T2) that followed with a significant drop at the day 28 (T3); the maximum ACP activity was reported between 3 and 6 weeks in Insoft et al. study. These differences could be due to several different variables including small sample size of named study which restricts the ability to perform statistical analysis on the longitudinal data (3 cases), the applied force direction (buccolingually) and also lower force level (100g) compared to the present study (approximately 250g). [14] Moreover, in the present study, ACP activity was much greater in distal sites in DCs compared with the mesial sites of all groups at T2. However, this difference was statistically significant only at CC group at this time point. This

Conclusion
Alterations of ALP and ACP activities in human GCF during orthodontic tooth movement may reflect the metabolic changes in periodontium. Although, changes in ACP activity in GCF may be considered to be an indicator of tissue modification during orthodontic tooth movement in human, it appears not to be sensitive as ALP in distinguishing between tension and compression sites after the treatment. Since other factors such as different clinical conditions may influence the enzymatic activities, the ALP and ACP of human GCF should be considered as reliable biomarkers of tissue responses to orthodontic tooth movement, only when the oral hygiene is kept under control.